Homocysteine (Hcy) is the immediate precursor of the amino acid, methionine. In humans, blood concentrations of Hcy may become elevated as a result of deficiency in folate, vitamin B6 or vitamin B12 and has recently been identified as a putative risk factor for a number of age-associated disease states including arteriosclerosis, myocardial infarction, arterial occlusive disease, Alzheimer's disease and neural tube defects. A specific role for Hcy or any of its metabolites, such as S-adenosyl Hcy (SAdy) or Hcy thiolactone, in these conditions has not yet been firmly established. Particularly absent is a description of the effects of elevated Hcy levels on immune function. Several studies have examined the effects of Hcy on monocyte, neutrophil and B cell function, inflammation and chemokine production; however, little is known about the Hcy effects on T lymphocytes. Our initial studies revealed that treatment of resting and activated human and murine T cells with L-Hcy resulted in a dose-dependent increase in apoptotic cell death. L or D,L Hcy was more potent than Hcy thiolactone in this respect, while SAdy was found to be significantly less active. We also found that the pro-apoptotic effects of Hcy were abrogated with the addition of serum, pan-caspase inhibitors, and poly-ADP-ribose polymerase (PARP) inhibitors to the cell cultures. These results suggest that D,L-Hcy, like other apoptotic stressors, leads to the activation of the caspase cascade and eventually to the cleavage of the key cellular proteins, like PARP, the release of mitochondrial cytochrome c, DNA fragmentation, and eventually leading to the typical morphological changes observed in cells undergoing apoptosis. We have also found that Hcy-mediated apoptosis is inhibited by the phosphatase inhibitor, sodium orthovanadate, the intracellular calcium chelator, BAPTA-AM, and the protein synthesis inhibitor, cycloheximide. Moreover, we have also found that Hcy appears to potentiate cellular death induced by a number of other established apoptotic signals including activation-induced cell death (AICD), heat shock, and Fas ligand- and HIV-mediated T cell death. Finally, we have also observed that in vivo infusion of D,L Hcy but not SAdy induces thymic involution with a dramatic reduction in cell numbers and thymic weights. The specific role of Hcy in age- or disease-associated involution remains to be defined. In addition to the pro-apoptotic effects of Hcy, stimulation of mononuclear cells or highly-purified human or murine T cells with immobilized anti-CD3 mAb in the presence of Hcy or thiolactone but not SAdy resulted in a significant increase in several type 1 cytokines including IL-2, IFN-gamma, TNF-alpha and IL-10 but not the type 2 cytokines, IL-4 or IL-5. These effects were also observed upon in vivo infusion of Hcy and within folate deficient mouse models. A more detailed examination of the Hcy effects on T cell activation revealed that this type 1 cytokine production profile is partially mediated through the production of IL-18, IL-12, and nitric oxide. Moreover, D,L-Hcy but not SAdy treatment also resulted in the demethylation of the IFN-gamma promoter permitting the binding of phosphorylated CREB. Given the potent effects of Hcy on methylation, these results may not seem surprising; however, a more detailed analysis of the direct induction of methyltransferase activity, DNA or protein methylation or indirect effects through other mediators and signals needs to be defined. The precise mechanism involved in the generation of these cytokines is currently under investigation but we believe the Hcy effect is being mediated, in part, by specific stress-associated signals post Hcy treatment. Detailed cytokine promoter analysis is currently underway to address the effects of Hcy on transcription factor induction and activation. Overall, Hcy appears to exert a number of differential effects on immune cells, which may alter immune function in the circulation and tissue microenvironment with age and inflammatory and/or autoimmune disease pathology. A greater understanding of the potential modulatory effects of Hcy and its metabolites on immune function may result in the development of potential therapeutic strategies to control and optimize immune responses with age, AIDS and in various age-associated disease states.